Figure 5.

NOTCH1 and CEBPδ are targets of the H9SB repressive complex. (A) The Venn diagram shows the overlap between genes commonly dysregulated upon HOXA9 and SAFB perturbation in MOLM13 cells (green) and genes that are linked to H9SB co-occupied genomic regions in primary AML cells and MOLM13 cell line (gray). A list of these genes is provided in supplemental Table 4. Hypergeometric test P < 2.115e-56. (B) GO analyses for target genes responded to HOXA9 or SAFB perturbation in MOLM13 cells and are common in primary AML cells and MOLM13 cells. The gene ratio is plotted on the x-axis. The colors on the bar represent the significant P value. A bar that represents the color code is shown on the right side of the plot. (C) Genome browser tracks demonstrate the enrichment of HOXA9 (blue) and SAFB (red) at the representative NOTCH1 locus in the Hg38 genome obtained from CUT&RUN sequencing in MOLM13 cells. The lower 2 tracks show the RNA-seq data and the expression of NOTCH1 to be upregulated in HOXA9-Cr (overlain blue) and SAFB-Cr (overlain red) compared with that in the NT control (gray). (D) Apoptosis in MOLM13-NOTCH1-ICN or MOLM13-MSCV-control cells was measured using annexin V– and 7AAD-positive cells by flow cytometry. Plots are representative of 3 biological independent replicates. (E) Competition assay between green fluorescent protein (GFP)–positive NOTCH1-ICN or murine stem cell virus (MSCV) control vector expressing MOLM13 cells. Equal numbers of GFP-positive cells were seeded at day 0, and the GFP percentage was measured 3 and 5 days later via flow cytometry. The values were normalized to the average percent from EV control and plotted as a line graph. Data shown are the averages of 3 biological replicates ± SD. (F) Colony-forming assay of GFP positive MOLM13-NOTCH1-ICN or MOLM13-MSCV-control cells. The bar graph shows the average value of 3 independent experiments ± SD. (G) Gene set enrichment analysis for H9SB commonly regulated genes (n = 2440) enriches for NOTCH signature. (H) Genome browser track representation of another exemplar locus (CEBPδ). Track details are as described for panel C. (I) Western blot showing the expression of CEBPδ in MOLM13 cells upon doxycycline treatment for 2 days. β-Tubulin was used as a loading control. (J) Cumulative cell growth of MOLM13 cells in CEBPδ overexpressing cells ± doxycycline. The experiment shown here is an average of 3 independent replicates obtained from 2 independent clones. Error bars represent ± SD. Statistical significance was calculated using 2-way ANOVA with multiple comparisons (95% CI) against EV-Dox, (EV-Dox-CEBPD-Dox at day 11, ∗∗∗∗P < .0001; EV-No Dox-CEBPD-No Dox at day 11, ∗∗P = .009). (K) Colony-forming assay of MOLM13-CEBPδ or MOLM13-control cells ± doxycycline. Photomicrographs showing representative plates (top). The scatter plot shows 3 independent experiments in duplicates, showing median ± SD (bottom). Statistical significance was calculated using 2-way ANOVA with multiple comparisons (95% CI) EV-Dox-CEBPD-Dox, ∗∗∗∗P < .0001. (L) Correlation between HOXA9 and NOTCH1 mRNA expression in human AML primary samples (n = 165, TCGA data set). (M) Correlation between HOXA9 and CEBPδ mRNA expression in human AML primary samples (n = 165, TCGA data set).